During development of biological drugs such as monoclonal antibodies (mAbs), it is important to establish comparability of key physicochemical properties, including higher-order structure (HOS), following changes and/or improvements to the manufacturing process. NMR spectroscopy is a non-destructive and data-rich analytical method that can report on higher-order structure, as well as dynamics, of macromolecules in solution at atomic resolution. Recent advances in NMR acquisition and analysis, including non-uniform sampling schemes, have allowed for the collection of highly reproducible fingerprint spectra of therapeutic mAbs. However, certain challenges in using NMR to analyze GMP-produced, natural abundance mAb samples remain, including the difficulty of making accurate peak assignments to get site-specific information. Here we show several different phase-appropriate applications of NMR spectroscopy in characterizing therapeutic mAb comparability and process consistency, using unbiased statistical approaches to obviate the need for residue-specific assignments. Computational comparisons using ECHOS-NMR and PCA of 1D and 2D NMR spectra establish comparability between different manufacturing processes, as well as intra-process consistency, while revealing small differences in HOS. Additionally, we demonstrate initial steps toward assignment of protein and glycan-derived signals allowing for preliminary quantitative and qualitative structure-function analyses of critical quality attributes.
